Bacteriological Survey of the Frozen Prepared Foods Industry: II. Frozen Breaded Raw Shrimp

During inspection of 21 frozen breaded raw shrimp processors, 861 finished product units (retail packages) and 778 line samples were collected and analyzed bacteriologically. The bacteriological results for the finished product samples collected in plants with poor and good sanitary controls are presented and compared. The samples consisted of 4 to 38 units (retail packages) of the finished product as offered for sale by the processors. Frozen raw breaded shrimp collected from plants operating under good conditions of sanitation had the following bacterial contents: 85% of the samples had an average (geometric) aerobic plate count of less than 1,000,000 per gram; all of the samples had an average (geometric) most probable number of less than 1,000 coliforms per g; 81% of the samples contained less than 20% Escherichia coli-positive units; 57% of the total number of units in 0.1-g portions were negative for coagulase-positive staphylococci, and 95% of the units had less than 1,000 coagulase-positive staphylococci per gram.     

Bacteriological Survey of the Frozen Prepared Foods Industry: I. Frozen Cream-Type Pies

During Food and Drug Administration inspections of 12 imitation cream pie producers, 453 finished product samples and 350 line samples were collected and analyzed bacteriologically. Sanitary conditions in the plants varied from good to poor and, in general, were reflected in the bacteriological results. The survey revealed that, in the great majority of cases, frozen imitation-cream pies produced in plants operating under good conditions of sanitation had the following bacteriological content: (i) a most probable number (MPN) of fewer than three Escherichia coli cells per gram (i.e., absent from all tubes in the methodology employed), (ii) an average MPN of fewer than 50 coliforms per gram (10 or more pies), (iii) the absence of coagulase-positive staphylococci in 0.1-g portions, and (iv) an aerobic plate count of fewer than 25,000 per gram (geometric mean of 10 or more pies).     

Bacteriological Survey of the Frozen Prepared Foods Industry: IV. Frozen Breaded Fish

During sanitation-inspections of 23 breaded-fish processors, 573 finished product units and 604 line samples were collected and analyzed bacteriologically. Data for finished product units processed on the same data are presented in groups; most of the groups consisted of 10 units. Groups of frozen, raw-breaded fish produced under good and poor conditions of sanitation were studied. No more than 20% of the units in each group produced under good conditions of sanitation were positive for Escherichia coli or coagulase-positive staphylococci. Groups of frozen, fried-breaded fish produced under good and poor sanitary conditions were also studied. No more than 10% of the units in each good-sanitation group were positive for E. coli or coagulase-positive staphylococci. However, approximately 30% of the groups processed under poor sanitary conditions also did not exceed these viable bacterial counts because of the lethal effect of the terminal fry to which the product is subjected. It was found that line samples collected from the processing lines reflected the sanitary conditions of the plant and provided bacteriological support for inspectional evidence of plant insanitation.     

Bacteriological Survey of the Blue Crab Industry

During sanitation inspections of 46 crabmeat processing plants on the Atlantic and Gulf Coasts, 487 samples of whole crabs immediately after cooking, cooked crabs after cooling, backed or washed (or both) crab bodies and whole crab claws, as well as 1,506 retail units of finished product were collected and analyzed bacteriologically. The 1,506 retail units (1-lb [373.24-g] cans) included 518 cans of regular (special) meat, 487 cans of claw meat, and 501 cans of lump meat. Statistical analyses showed that crabmeat from plants in Mississippi, Louisiana, and Texas had higher counts in 19 of 24 cases for the four bacteriological indices than crabmeat from plants located along the Atlantic Coast and the Gulf Coast of Florida. Aerobic plate counts of retail units collected from a previous day's production were significantly higher than those collected on the day of inspection. Regular crabmeat had consistently higher aerobic plate counts than claw or lump meat. When the product was handled expeditiously under good sanitary conditions, the bacteriological results were significantly better than the results from plants operating under poor sanitary conditions. Crabmeat produced in plants operating under good sanitary conditions had the following bacteriological content: (i) coliform organisms average most-probable-number values (geometric) of less than 20 per g; (ii) no Escherichia coli; (iii) coagulase-positive staphylococci average most-probable-number values (geometric) of less than 30 per g in 93% of the plants; (iv) aerobic plate count average values (geometric) of less than 100,000 per g in 93% of the plants, with the counts from 85% of these plants below 50,000 per g.     

EVALUATION OF THE PETRIFILMTM AND REDIGELTM AS RAPID METHODS TO THE STANDARD PLATE COUNT METHOD FOR THE ENUMERATION OF PROCESSED MEATS AND ENVIRONMENTAL SAMPLES

Aerobic plate counts (APC) are used by the food industry to help determine the sanitary state of equipment and bacterial load of finished product. Conventional aerobic count methods for processed meats require approximately 72 h incubation before results are available. Food processing plants with in-house analytical laboratories require methods that are simple, reliable and rapid. New test methods using Petrifilm^TM (PF) and Redigel^TM (RG) have been developed for determining APC. These methods are faster and easier than culturing in a standard agar medium. A variety of raw, cooked; cured and noncured meat products, cooling brine and environmental surface swabs were collected and analyzed for APC using PF, RG and plate count agar (PCA). Data analyses from over 200 samples indicated that the sensitivity of rapid testing methods for aerobic bacteria can vary depending on sample type.     

Elimination of viruses and indicator bacteria at each step of treatment during preparation of drinking water at seven water treatment plants

Seven drinking water treatment plants were sampled twice a month for 12 months to evaluate the removal of indicator bacteria and cytopathogenic enteric viruses. Samples were obtained at each level of treatment: raw water, postchlorination, postsedimentation, postfiltration, postozonation, and finished (tap) water. Raw water quality was usually poor, with total coliform counts exceeding 105 to 106 CFU/liter and the average virus count in raw water of 3.3 most probable number of cytopathogenic units (MPNCU)/liter; several samples contained more than 100 MPNCU/liter. All plants distributed finished water that was essentially free of indicator bacteria as judged by analysis of 1 liter for total coliforms, fecal coliforms, fecal streptococci, coagulase-positive staphylococci, and Pseudomonas aeruginosa. The total plate counts at 20 and 35 degrees C were also evaluated as a measure of the total microbial population and were usually very low. Viruses were detected in 7% (11 of 155) of the finished water samples (1,000 liters) at an average density of 0.0006 MPNCU/liter the highest virus density measured being 0.2 MPNCU/liter. The average cumulative virus reduction was 95.15% after sedimentation and 99.97% after filtration and did not significantly decrease after ozonation or final chlorination. The viruses isolated from treated waters were all enteroviruses: poliovirus types 1, 2, and 3, coxsackievirus types B3, B4, and B5, echovirus type 7, and untyped picornaviruses.     

Elimination of viruses and indicator bacteria at each step of treatment during preparation of drinking water at seven water treatment plants.

Seven drinking water treatment plants were sampled twice a month for 12 months to evaluate the removal of indicator bacteria and cytopathogenic enteric viruses. Samples were obtained at each level of treatment: raw water, postchlorination, postsedimentation, postfiltration, postozonation, and finished (tap) water. Raw water quality was usually poor, with total coliform counts exceeding 105 to 106 CFU/liter and the average virus count in raw water of 3.3 most probable number of cytopathogenic units (MPNCU)/liter; several samples contained more than 100 MPNCU/liter. All plants distributed finished water that was essentially free of indicator bacteria as judged by analysis of 1 liter for total coliforms, fecal coliforms, fecal streptococci, coagulase-positive staphylococci, and Pseudomonas aeruginosa. The total plate counts at 20 and 35 degrees C were also evaluated as a measure of the total microbial population and were usually very low. Viruses were detected in 7% (11 of 155) of the finished water samples (1,000 liters) at an average density of 0.0006 MPNCU/liter the highest virus density measured being 0.2 MPNCU/liter. The average cumulative virus reduction was 95.15% after sedimentation and 99.97% after filtration and did not significantly decrease after ozonation or final chlorination. The viruses isolated from treated waters were all enteroviruses: poliovirus types 1, 2, and 3, coxsackievirus types B3, B4, and B5, echovirus type 7, and untyped picornaviruses.     

Relationships between the density of different indicator organisms on sheep and beef carcasses and in frozen beef and sheep meat

AIM: To describe the relationship between the concentration of different indicator bacteria in red meat. METHODS AND RESULTS: Enumeration data for aerobic plate count (APC), Enterobacteriaceae, coliforms and Escherichia coli biotype I were analysed from an Australia-wide survey of beef carcasses, sheep carcasses, frozen beef and frozen sheep meat. In all commodities, there was only low-to-moderate rank correlation (0.16-0.47) between concentration of APC and concentration of each Gram-negative indicator. Rank correlations between counts of Gram-negative indicators were much higher (0.47-0.92) especially when nondetections were excluded from analysis (0.78-0.94). Receiver-operator characteristics analysis showed that detection of coliforms can predict the presence of E. coli biotype I with almost 100% sensitivity but fails to predict absence in 2.7-8.5% of samples not containing E. coli biotype I. CONCLUSIONS: Enumeration of coliforms is a useful adjunct to enumeration of E. coli biotype I or Enterobacteriaceae in red meat. The density of coliforms or Enterobacteriaceae can be used to predict the presence or absence of E. coli biotype I, although when the latter is at low prevalence errors in positive test prediction can be large. SIGNIFICANCE AND IMPACT OF THE STUDY: A quantitative basis is provided for comparing the concentration of different indicator bacteria measured in the production, regulation and trade of red meat.     

Sanitary quality of marine sediments and sands from an Italian beach

Microbiological surveys were carried out on marine sands and sediments collected at a sandy beach along a coastal area close to Rome, Italy. Low densities of faecal indicator bacteria were recovered, and among them enterococci outnumbered the coliforms. The group of staphylococci was in a fairly constant concentration throughout the period of sampling. Some statistically significant correlations were calculated between yeasts and moulds, Escherichia coli and enterococci and between the latter and Clostridium spores. The data obtained could be a reference point for further studies.     

Detection of white spot syndrome virus (WSSV) of shrimp by means of monoclonal antibodies (MAbs) specific to an envelope protein (28 kDa)

The vp28 gene encoding an envelope protein (28 kDa) of white spot syndrome virus (WSSV) was amplified from WSSV-infected tiger shrimp that originated from Malaysia. Recombinant VP28 protein (r-28) was expressed in Escherichia coli and used as an antigen for preparation of monoclonal antibodies (MAbs). Three murine MAbs (6F6, 6H4 and 9C10) that were screened by r-28 antigen-based enzyme-linked immunosorbent assay (ELISA) were also able to recognize viral VP28 protein as well as r-28 on Western blot. Three non-overlapping epitopes of VP28 protein were determined using the MAbs in competitive ELISA; thus, an antigen-capture ELISA (Ac-ELISA) was developed by virtue of these MAbs. Ac-ELISA can differentiate WSSV-infected shrimp from uninfected shrimp and was further confirmed by a polymerase chain reaction (PCR) and Western blot. Approximately 400 pg of purified WSSV sample and 20 pg of r-28 could be detected by Ac-ELISA, which is comparable in sensitivity to PCR assay but more sensitive than Western blot in the detection of purified virus. Hemolymph and tissue homogenate samples collected from a shrimp farm in Malaysia during December 2000 and July 2001 were also detected by Ac-ELISA and PCR with corroborating results.     

Isolation of fecal coliforms from pristine sites in a tropical rain forest.

Samples collected from water accumulated in leaf axilae of bromeliads (epiphytic flora) in a tropical rain forest were found to harbor fecal coliforms. Random identification of fecal coliform-positive isolates demonstrated the presence of Escherichia coli. This bacterium was also isolated from bromeliad leaf surfaces. These data indicate that E. coli may be part of the phyllosphere microflora and not simply a transient bacterium of this habitat. The isolation of fecal coliforms from these sites was unexpected and raises questions as to the validity of using fecal coliforms as indicators of biological water quality in the tropics.     

Fluorogenic assay for rapid detection of Escherichia coli in food.

An assay procedure to screen for Escherichia coli in foods by using 4-methylumbelliferyl-beta-D-glucuronide (MUG) incorporated into lauryl tryptose (LST) broth was evaluated. The beta-glucuronidase produced by E. coli cleaves the MUG substrate to yield a fluorescent end product. E. coli-negative samples can be identified by lack of fluorescence in LST-MUG within 24 h. MUG was not inhibitory to coliforms and E. coli. Over 1,400 food and dairy samples were tested to compare the standard three-tube most-probable-number procedure with the MUG-containing or non-MUG-containing LST procedure. LST-MUG testing detected a greater number of E. coli, with a lower false-positive rate (1.4%) and in a shorter time, than did the standard procedure. All false-positive results in the LST-MUG testing were attributable to beta-glucuronidase-producing staphylococci. No false-negative result was encountered. Use of MUG in LST broth obviates the EC broth step, allowing a 2.5-day procedure to a completed E. coli test versus the present 4- to 6-day standard most-probable-number method.     

Fluorogenic assay for rapid detection of Escherichia coli in food

An assay procedure to screen for Escherichia coli in foods by using 4-methylumbelliferyl-beta-D-glucuronide (MUG) incorporated into lauryl tryptose (LST) broth was evaluated. The beta-glucuronidase produced by E. coli cleaves the MUG substrate to yield a fluorescent end product. E. coli-negative samples can be identified by lack of fluorescence in LST-MUG within 24 h. MUG was not inhibitory to coliforms and E. coli. Over 1,400 food and dairy samples were tested to compare the standard three-tube most-probable-number procedure with the MUG-containing or non-MUG-containing LST procedure. LST-MUG testing detected a greater number of E. coli, with a lower false-positive rate (1.4%) and in a shorter time, than did the standard procedure. All false-positive results in the LST-MUG testing were attributable to beta-glucuronidase-producing staphylococci. No false-negative result was encountered. Use of MUG in LST broth obviates the EC broth step, allowing a 2.5-day procedure to a completed E. coli test versus the present 4- to 6-day standard most-probable-number method.     

Changes in microbial population during fermentation of tropical freshwater fish viscera

Freshwater fish viscera (FV) was homogenized, mixed with 10% (w/w of FV) molasses and 0, 2 or 4% salt and allowed to ferment at ambient temperature (26·2°C) under microaerophilic conditions. The results revealed a reduction in total viable count and the number of spores, coliforms, Escherichia coli , staphylococci and enterococci and an increase in yeasts and moulds and lactic acid bacteria during fermentation. Coliforms and E. coli were found to be absent after 6 d and enterococci on 8th day. The presence of salt resulted in a marginally lower number of all organisms except yeasts, moulds and lactic acid bacteria. Inclusion of either 0·5% propionic acid, 0·3% calcium propionate or 0·1% sorbic acid suppressed growth of yeasts and moulds with propionic acid being the most effective. The study indicated that a microbiologically stable product could be prepared by ensiling fish viscera with 10% molasses and 0·5% propionic acid.     

Isolation of fecal coliforms from pristine sites in a tropical rain forest

Samples collected from water accumulated in leaf axilae of bromeliads (epiphytic flora) in a tropical rain forest were found to harbor fecal coliforms. Random identification of fecal coliform-positive isolates demonstrated the presence of Escherichia coli. This bacterium was also isolated from bromeliad leaf surfaces. These data indicate that E. coli may be part of the phyllosphere microflora and not simply a transient bacterium of this habitat. The isolation of fecal coliforms from these sites was unexpected and raises questions as to the validity of using fecal coliforms as indicators of biological water quality in the tropics.     

Molecular studies on the ecology of Listeria monocytogenes in the smoked fish processing industry

We have applied molecular approaches, including PCR-based detection strategies and DNA fingerprinting methods, to study the ecology of Listeria monocytogenes in food processing environments. A total of 531 samples, including raw fish, fish during the cold-smoking process, finished product, and environmental samples, were collected from three smoked fish processing facilities during five visits to each facility. A total of 95 (17.9%) of the samples tested positive for L. monocytogenes using a commercial PCR system (BAX for Screening/Listeria monocytogenes), including 57 (27.7%) environmental samples (n = 206), 8 (7.8%) raw material samples (n = 102), 23 (18.1%) samples from fish in various stages of processing(n = 127), and 7 (7.3%) finished product samples (n = 96). L. monocytogenes was isolated from 85 samples (16.0%) using culture methods. Used in conjunction with a 48-h enrichment in Listeria Enrichment Broth, the PCR system had a sensitivity of 91.8% and a specificity of 96.2%. To track the origin and spread of L. monocytogenes, isolates were fingerprinted by automated ribotyping. Fifteen different ribotypes were identified among 85 isolates tested. Ribotyping data established possible contamination patterns, implicating raw materials and the processing environment as potential sources of finished product contamination. Analysis of the distribution of ribotypes revealed that each processing facility had a unique contamination pattern and that specific ribotypes persisted in the environments of two facilities over time (P < or = 0.0006). We conclude that application of molecular approaches can provide critical information on the ecology of different L. monocytogenes strains in food processing environments. This information can be used to develop practical recommendations for improved control of this important food-borne pathogen in the food industry.     

Coagulase-negative staphylococci and coliform bacteria associated with mechanical defeathering of poultry carcasses

Coagulase-negative staphylococci and coliform bacteria were isolated from defeathering machines and carcasses at a commercial poultry processing plant Initially, the predominant staphylococci on carcasses were Staphylococcus xylosus and Staph simulans but during defeathering, these organisms were replaced by Staph. sciuri. Since Staph sciuri predominated in the defeathering machines, the machinery appeared to be responsible for contaminating carcasses.
Among the coliform bacteria, Escherichia coli was the principal organism isolated from both carcasses and defeathering machines However use of a biotyping scheme revealed no clear change in the pattern of carcass contamination during defeathering and it was concluded that E. coli is unlikely to colonize the machines.     

Evaluation of beta-glucuronidase assay for the detection of Escherichia coli from environmental waters.

The new United States Drinking Water Regulations state that water systems must analyze for Escherichia coli or fecal coliforms on any routine or repeat sample that is positive for total coliforms. The proposed methods for the detection of E. coli are based on beta-glucuronidase activity, using the fluorogenic substrate 4-methylumbelliferyl beta-D-glucuronide (MUG). This study was conducted to determine whether beta-glucuronidase negative E. coli were present in significant numbers in environmental waters. Two hundred and forty E. coli cultures were isolated from 12 water samples collected from different environmental sources. beta-glucuronidase activity was determined using lauryl tryptose broth with MUG, EC broth with MUG, and the Autoanalysis Colilert (AC) procedure. The isolates were also evaluated by the standard EC broth gas fermentation method for fecal coliforms. The results confirm that assaying for the enzyme beta-glucuronidase utilizing the MUG substrate is an accurate method for the detection of E. coli in environmental waters.     

A study of the microbiological status of Irish farmhouse cheeses with emphasis on selected pathogenic and spoilage micro-organisms

H.M. COVENEY, G.F. FITZGERALD AND C. DALY. 1994. Ninety-six 25 g samples from 25 Irish farmhouse cheeses, two Irish non-farmhouse cheeses and four foreign cheeses were evaluated for the presence of a variety of micro-organisms, namely, coliforms, faecal streptococci, Staphylococcus aureus , yeasts, moulds, salmonellas and shigellas. Seventeen cheeses, i.e. the soft and semi-soft types, were examined for Listeria monocytogenes. Most of the farmhouse cheeses are currently manufactured from raw milk, but some producers now use heat-treated milk. The incidence of coliforms and faecal coliforms was higher in soft, semi-soft and semi-hard cheeses than in hard types. High levels of contamination by faecal streptococci and non-pathogenic (coagulase-negative) Staph. aureus prevailed in a high proportion of the cheeses. Pathogenic (coagulase-positive) staphylococci, however, were also isolated from 50% of the cheeses, some of which were manufactured from pasteurized milk. Yeasts were found mainly in unpasteurized varieties, especially in the category of soft cheeses. Moulds were isolated from five non-mould-ripened cheeses, as well as from mould-ripened varieties. Salmonellas, shigellas and Listeria monocytogenes were not detected after direct enrichment.     

A non-destructive method based on the polymerase chain reaction for detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp

Current methods to detect hepatopancreatic parvovirus (HPV) infection of penaeid shrimp depend on invasive techniques that require dissecting the organs infected by this virus. However, sacrificing valuable stocks in order to determine their HPV status can be a drawback in the case of breeding programs. A method was developed for HPV detection by applying a polymerase chain reaction (PCR) assay to fecal samples collected from live HPV-infected shrimp Penaeus chinensis. A pair of PCR primers, 1120F/1120R, which amplify a 592 base pair (bp) region from the virus genome, was designed from previously known HPV sequence information (HPV clone HPV8). PCR amplification with these primers generated a product of the expected size directly from the crude feces of HPV-infected shrimp but not from the feces of specific pathogen-free (SPF) shrimp. The HPV origin of the amplified product was validated by means of an in situ hybridization assay where the product of the amplification, labeled with digoxigenin (DIG)-11-dUTP, showed an intense reaction within hepatopancreatic cells displaying characteristic HPV lesions on HPV-infected shrimp. No reaction to this probe was observed when reacted in situ with sections of the hepatopancreas of SPF specimens or to sections of shrimp infected by the infectious hypodermal and hematopoietic necrosis virus (IHHNV), another parvovirus of penaeid shrimp. These primers were tested for specificity against homologous and nonhomologous viruses and no product was amplified. A fragment of the expected size was obtained only when purified HPV or purified HPV8 plasmid was used as template DNA. Under optimized conditions, these primers detected as little as 1 fg of purified HPV8 plasmid DNA, equivalent to approximately 300 HPV particles. Analysis of fecal samples by PCR may prove useful for non-lethal screening of valuable shrimp of unknown HPV status. This same strategy also might be used for detection of other enteric viruses that infect penaeid shrimp.